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Acknowledgements

Materials

References

Immunology and Infection

Experimental Infection with Listeria monocytogenes as a Model for Studying Host Interferon-γ Responses

Published: November 16th, 2016

DOI:

10.3791/54554

1Department of Immunology, University of Toronto, 2Toronto General Research Institute, University Health Network, 3Women's College Research Institute
* These authors contributed equally

This protocol describes how to inoculate C57BL/6J mice with the EGD strain of Listeria monocytogenes (L. monocytogenes) and to measure interferon-γ (IFN-γ) responses by natural killer (NK) cells, natural killer T (NKT) cells, and adaptive T lymphocytes post-infection. This protocol also describes how to conduct survival studies in mice after infection with a modified LD50 dose of the pathogen.

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been highly informative on the role of innate and adaptive immune cells and specific cytokines in host immunity against intracellular pathogens. Production of IFN-γ by innate cells during sublethal infection with L. monocytogenes is important for activating macrophages and early control of the pathogen1-3. In addition, IFN-γ production by adaptive memory lymphocytes is important for priming the activation of innate cells upon reinfection4. The L. monocytogenes infection model thus serves as a great tool for investigating whether new therapies that are designed to increase IFN-γ production have an impact on IFN-γ responses in vivo and have productive biological effects such as increasing bacterial clearance or improving mouse survival from infection. Described here is a basic protocol for how to conduct intraperitoneal infections of C57BL/6J mice with the EGD strain of L. monocytogenes and to measure IFN-γ production by NK cells, NKT cells, and adaptive lymphocytes by flow cytometry. In addition, procedures are described to: (1) grow and prepare the bacteria for inoculation, (2) measure bacterial load in the spleen and liver, and (3) measure animal survival to endpoints. Representative data are also provided to illustrate how this infection model can be used to test the effect of specific agents on IFN-γ responses to L. monocytogenes and survival of mice from this infection.

IFN-γ is a cytokine that is crucial for mediating immunity against intracellular pathogens and for controlling tumor growth5. The importance of this cytokine in bacterial resistance is evident in the observation that humans with mutations in the IFN-γ signaling pathway are highly susceptible to infection with mycobacteria and salmonellae6. Similarly, mice deficient in either IFN-γ or the IFN-γ receptor exhibit defects in resistance to mycobacteria7-9 and other intracellular pathogens including L. monocytogenes10,11, Leishmania major12, Salmonella typhimurium13

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Safety Statement

This protocol describes infection of mice with live L. monocytogenes. The pathogen is handled safely under BSL2 conditions by trained personnel who are not immunocompromised. Immunocompromised people include pregnant women, the elderly and individuals who are HIV-infected or have chronic conditions that require treatment with immunosuppressive therapy. Personnel should don a protective lab coat or gown, gloves, mask, and eye protection while handling infected samples. T.......

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Figure 3 presents some typical flow cytometry staining of IFN-γ in splenic NK and NKT cells at 24 hr post-infection with 105 CFU of the pathogen. This figure also illustrates the gating strategy for the staining panel described in Table 2. Figure 4 shows some representative data that were obtained in one experiment where male mice were treated with the PPARα antagonist IS001 or vehicle control, infected with 105<.......

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Here we describe a protocol of how to carry out a basic experimental infection with the EGD strain of L. monocytogenes25 in male or female C57BL/6J mice. This protocol was set up for the purpose of studying the effect of a novel small molecule IS001 on IFN-γ production by innate and adaptive lymphocytes in vivo31. By monitoring bacterial clearance and survival post-infection, insights were gained into how these changes in IFN-γ impacted the host's ability to control t.......

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Development of this protocol was supported by an operating grant from CIHR (MOP97807) to SED.

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Name Company Catalog Number Comments
Brain Heart Infusion Broth, Modified BD 299070 any brand should be appropriate
Agar BD 214010 any brand should be appropriate
Triton X-100 Sigma-Aldrich X100 any brand should be appropriate
1xPBS Sigma D8537 any brand should be appropriate
TissueLyser II Qiagen 85300 any brand should be appropriate
Ammonium Chloride (NH3Cl) any brand should be appropriate
KHCO3 any brand should be appropriate
Na2EDTA any brand should be appropriate
RPMI 1640 Gibco 22400089 any brand should be appropriate
Fetal Bovine Serum  Gibco 12483 Before use, heat-inactivate at 56 °C for 30 min
L-glutamine Gibco 25030 any brand should be appropriate
Non-essential amino acids Gibco 11140 any brand should be appropriate
Penicillin/Streptomycin Gibco 15140 any brand should be appropriate
GolgiStop Protein Transport Inhibitor (containing Monensin) BD 554724 Use 4 μl in 6 ml cell culture
16% Paraformaledehye Electron Microscopy Sciences 15710 Dilute to 4% PFA in ddH20 or 1xPBS
10 x Perm/Wash buffer BD 554723 Dilute 10x in ddH20
Fc block, Anti-Mouse CD16/CD32 Purified eBioscience 14-0161 Dilute 1:50
Fixable Viability Dye eFluor 506 eBioscience 65-0866 Dilute 1:1000 (we have also used viability dyes from Molecular Probes)
anti-Mouse CD4-PE-Cy5 (GK1.5) eBioscience 15-0041 Manufacturer recommends a certain test size; however this should be titrated before use.
anti-Mouse CD8-FITC (53-6.7) eBioscience 11-0081 Manufacturer recommends a certain test size; however this should be titrated before use.
PBS57/mCD1d tetramer-APC NIH Tetramer Core Facility N/A Obtained as a gift from the facility
anti-Mouse TCRβ-PE-Cy7 (H56-597) eBioscience 25-5961 Manufacturer recommends a certain test size; however this should be titrated before use.
anti-Mouse NKp46-APC-eFluor780 (29A1.4) eBioscience 47-3351 Manufacturer recommends a certain test size; however this should be titrated before use.
anti-Mouse CD45 PE-Cyanine7 (30-F11) eBioscience 25-0451 Manufacturer recommends a certain test size; however this should be titrated before use.
anti-Mouse IFN gamma-PE (XMG1.2) eBioscience 12-7311 Manufacturer recommends a certain test size; however this should be titrated before use.
OneComp eBeads eBioscience 01-1111 Manufacturer recommends a certain test size; however this should be titrated before use.
Mouse IFN gamma ELISA kit eBioscience 88-7314 Used for measuring the interferon gamma in the culture supernatant
50 mL vented tubes for culture Used for culturing the bacteria, any brand should be appropriate
1.5 ml microcentrifuge tubes any brand should be appropriate
bacterial petri dishes any brand should be appropriate
2 ml cyrovials any brand should be appropriate
UV spectrometer any brand should be appropriate
safety engineered needles any brand should be appropriate
C57BL6/J Jackson laboratories Stock#000664 Order for arrival at 7 wks
Bleach For decontamination
70% Ethanol For decontamination
Glass beads any brand should be appropriate
Centrifuge rotor, buckets, bucket covers.
Microcentrifuge any brand should be appropriate
Sterile Glycerol any brand should be appropriate
Pipette Tips any brand should be appropriate
Pipette any brand should be appropriate
Surgical instruments any brand should be appropriate
70 micron strainers any brand should be appropriate
3 ml syringe any brand should be appropriate
Pipette gun any brand should be appropriate
Filtration Units any brand should be appropriate
Trypan Blue Dilute 1 to 9 in ddH20, any brand should be appropriate
Hemocytometer any brand should be appropriate
Round bottomed plates any brand should be appropriate
FACs tubes BD
BD LSR II BD Any flow cytometer could be used for acquisition that has an appropriate laser configuration and filter set to discriminate the fluorochormes
Flowjo software Treestar Used for data analysis. Other types of data analysis software will also be appropriate
Multichannel pipettor (0-300 µl) Eppendorf Used for washing cells and adding antibodies during flow cytometry staining
Acetic Acid Used for washing glass beads, any brand should be appropriate
Microbank Bacterial Preservation System Pro-lab Diagnositics Used as an alternative to glycerol stocks for long-term storage of bacteria

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